| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
In order to develop a method for structural characterization of DNA-binding proteins with multiple structural domains, chemical cross-linking and mass spectrometry analyses were carried out on a complex of a DNA-binding domain of transcription factor HSF3 and its target protein. After the observation of the molecular ion of the non-covalent protein-protein complex, cross-linking of two amino groups of Lys side chains were performed with BS^3 (bis(sulfosuccinimidyl) suberate). The cross-linked protein complex was isolated by SDS-PAGE, reduced and alkylated, and digested with trypsin in the gel. The digest was analyzed by MALDI-TOFMS and assignment of molecular masses to the sequence was carried out, resulting in the identification of linked Lys residues. With further improvement, this method is promising for the characterization of a protein with multiple structural domains, whose tertiary structure is difficult to be solved with X-ray crystallography or NMR.
Expression and purification
… More
of the full-length HSF3 and TFIIE, transcription factors with multiple structural domains, was carried out. Since the expression level of HSF3 was low with the initial system, several kinds of plasmid and host E. coli were examined. In the case of TFIIE, heterotetramer of two TFIIEα and β, there were difficulties in the expression level (TFIIEα) and in the stability during the purification process (TFIIEβ). The method mentioned above will be applied to these proteins after the establishment of their expression and purification systems.
Structural changes on a peptide by the interaction with phospholipids were investigated with hydrogen-deuterium exchange and Fourier-transform ion cyclotron resonance mass spectrometry. Melittin, a hemolytic peptide isolated from honeybee, was in quite random structure in the absence of lipids while it was in some stable structure in the presence of phospholipids. Different behavior was recognized according to the kinds of phospholipids. This method might be applicable to characterize the structure of membrane-bound proteins. Less
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